Dengue virus (DV) infection causes a spectrum of disease ranging from self-limited Dengue Fever to lifethreatening Dengue Hemorrhagic Fever/Dengue Shock Syndrome. The mechanisms of immune protection and pathogenesis are poorly understood, but DV nonstructural protein 1 (NS1) likely plays a key role. NS1 is present in the cytoplasm and on the surface of infected cells and is secreted in a soluble form (sNS1) that circulates in blood, where high sNS1 concentration correlates with increased disease severity. In vitro, sNS1 can activate complement, bind to infected and uninfected cells, and increase viral output from infected hepatoma cells. However, it is unclear if or how sNS1 affects viral dissemination or disease progression in vivo. Similarly, the role of antibodies (Abs) against NS1 is unclear due to the lack of appropriate in vivo models. Anti-NS1 Abs display protective activity against neurovirulent death in mice, but the effects of anti-NS1 Abs on DV infection of more relevant peripheral tissues have never been characterized. Nonetheless, NS1 is included in several DV vaccines under development, including that of our collaborator, Hawaii Biotech, Inc., and no test exists to evaluate the repertoire of Abs generated by either natural infection or vaccination. We have shown that DV infection of interferon receptor-deficient mice reproduces key features of human DV infection, including susceptibility to all four DV serotypes with relevant infection kinetics, appropriate tissue and cellular tropism, sNS1 circulation in the blood, thrombocytopenia correlated with viral load, and fatal vascular leak syndrome. We will use this mouse model to examine the functions of sNS1 and the effects of anti-NS1 Abs on DV infection. We will also use sera obtained from mice, prospective studies of dengue in Nicaragua, and dengue vaccine trials to generate in vitro assays to assess the repertoire of anti-NS1 Abs in infected mice, primary and secondary natural DV infections, and vaccine recipients. In Specific Aim 1, we will assess the localization and function of sNS1 during DV infection in mice and fatal human dengue cases. In Specific Aim 2, the effects of anti-NS1 Abs on peripheral DV infection in mice will be evaluated. We will measure the ability of both polyclonal Abs and MAbs against NS1 to protect against DV infection using both NS1 vaccination and MAbs against DV2 NS1, and we will screen for any detrimental in vivo effects of NS1 Abs. Finally, in Specific Aim 3, we will characterize the repertoire of anti-NS1 Abs generated by DV infection and vaccination and will develop in vitro tests to detect anti-NS1 Abs in human serum likely to have protective or pathogenic effects during subsequent DV infection. Together, these results will both elucidate mechanisms of DV pathogenesis and contribute to the development of a safe and effective dengue vaccine.